The immoderate use of different antibiotics during the last six decades caused the emergence and the dissemination of bacterial populations expressing resistance to these substances. Interactions between microbial resistance and antibacterial agents occur either directly by the development of resistance to the agent used, to agents of the same class, or in an indirect way by the selection of resistant organisms when patients are treated with antibiotics, when the environment is contaminated with antibiotics (hospital) or when antibacterial agents are applied in agriculture and animal husbandry. Antimicrobial selection as well as contagion are the decisive processes responsible for the global spread and linking of resistance genes.
There is increased concern that because of the widespread use of antibiotics and the continuous increase of bacterial resistance, the pharmaceutical industry may no longer be able to develop effective novel antibiotics sufficiently rapidly (JACK et al. 1995). This fact quickens interest in microbially produced antibacterial peptides. In recent years, more than fifty antibacterial peptides produced by lactic acid bacteria have been isolated (NES and HOLO, 2000). These so-called bacteriocins contain 20-60 amino acids, are inhibitory in a nanomolar range and cause membrane permeabilization and leakage of sensitive cells. They are secreted by some bacterial strains, which are thus adapted to compete against other microorganisms in the same environmental compartment (NAVARRO et al. 2000).
Only a few microbially produced substances are known to be effective against gram-positive as well as gram-negative bacteria (BLACKBURN et al. 1989; STEVENS et al. 1991; KALCHAYANAND et al. 1992). One promising substance of biogenic origin possessing a broad antimicrobial spectrum (JOHANSEN et al. 1995; JOHANSEN et al. 1997; HANSEN and GILL 2000) is protamine, a basic peptide (pI>10) consisting of 32 amino acids, of which 21 are arginine (ANDO et al. 1973). Protamine is found enriched in sperm, e.g. in salmon spermatozoan nuclei, compacting DNA and taking the position of histones during the maturation of the spermatids (LOUIE and DIXON, 1974). Its effect on gram-negative bacteria is reported to be lower than on gram-positive bacteria (IsLAM et al. 1984; MULHOLLAND and MELLERSH 1987; YANAGIMOTO 1992; JOHANSEN et al. 1995).
There is an extremely high degree of sequence conservation in the coding and 3′ untranslated regions of different rainbow trout protamine genes (AIKEN et al. 1983), showing cross-hybridization under less stringent conditions (SAKAI et al. 1981).
Protamine is also known for its antifungal activity (KAMAL et al. 1986).
The antimicrobial effect of protamine is supposed to be caused by its polycationic nature (HIRSCH 1958; JOHANSEN et al. 1995). A possible mode of action may consist in the binding of the peptide to the outer membrane, causing a malfunction and inducing channels into the membrane, as was proved for other cationic peptides (CHRISTENSEN et al. 1988; KAGAN et al. 1990).
It has been demonstrated that protamine causes a disruption of the cytoplasmatic membrane (JOHNSEN et al. 1997). It was furthermore shown that polycationic peptides like protamine can also activate autolysins, resulting in cell lysis, as well as inhibit phosphorylase activity (JOHANSEN et al. 1996). Protamine may also enter the cytoplasm, inhibiting genetic transformation (ANTHOI and POPESCU 1979). Protamine penetrates gram-negative bacteria and enhances the permeability of the outer membrane (VAARA and VAARA 1983; VAARA 1992). Furthermore, it deenergizes bacterial membranes and causes a reduction of cellular ATP content (ASPEDON and GROISMAN, 1996).
It has been shown that the antibacterial activity of protamine is dependent on the amount of bivalent cations stabilizing the outer membrane (JOHASEN et al. 1997) and the pH value (HANSEN and GILL 2000).
Protamine was shown to extend antibacterial effects against food-spoiling bacteria (JOHANSEN et al. 1996). Interestingly, STUMPE et al. (1998) found that OmpT, a protease from E. coli, is able to degrade protamine before it enters the bacterial cells. This fact has to be taken into consideration if protamine is used as a disinfectant or a replacement for antibiotics. It is of great importance that antibiotics are no longer to be permitted in the food industry after the end of 2003. Thus, new and effective substances have to be at disposal before that time.
WO 96/06532 discloses a protamine composition for killing or inhibiting microbial cells. The studies have been performed with protamine from Salmon (P-4005) obtained from Sigma Chemical Company (St. Louis, USA), dissolved in destined water, filter sterilized (0.2 μm) and used immediately after preparation. Besides protamine or protamine sulphate, the composition disclosed in WO 96/06532 additionally comprises a cell-wall degrading enzyme and/or an oxidoreductase and allegedly displays bactericidal, bacteriostatic, fungicidal and/or fungistatic properties. Accordingly and due to its broad range of activity against target organisms, protamine containing preparations have already been suggested for detergent and hard surface cleaning compositions and in methods for killing microbial cells present on a hard surface, for killing microbial cells or inhibiting growing microbial cells present on laundry, for killing microbial cells present on human or animal skin, mucous membranes, wounds, bruises or in the eye, as well as for the preservation of food, beverages, cosmetics, contact lens products, food ingredients or enzyme compositions.
Furthermore, protamine and suitable derivatives thereof display important physiological functions and have been suggested for medical use. For example, protamine has proven as a clinical heparin antagonist, e.g. to reduce post-operative bleeding, and is routinely administered after cardiac and vascular surgery to reverse the anticoagulant activity of heparin. In addition, protamine prolongs the adsorption of insulin, and is therefore combined with insulin to formulate protamine zinc insulin (PZI) and neutral protamine Hagedorn (NPH) insulin. Such formulations allow insulin-dependent diabetic patients to achieve euglycemia with less frequent insulin injections.
However, despite its universal use in clinical practice, current formulations of protamine are nevertheless toxic. Protamine toxicity ranges from mild hypotension to severe systemic vascular collapse requiring prompt intervention, or idiosyncratic fatal cardiac arrest. This drawback has been addressed in WO 00/55196 relating to low molecular weight (LMW) bioactive protamines and compositions, allegedly having reduced immunogenicity, antigenicity and/or toxicity compared to native protamine. According to the teaching of WO 00/55196, native salmine® (salmon) or clupeine® (herring) protamine commercially available from Sigma Chem. Co. is contacted with a proteolytic composition to generate LMW protamine fragments which can subsequently be used to select appropriate fragments, polypeptides, species of fractions of interest. However, WO 00/55196. does not provide any sequence information for the LMW protamine fragments.
It has thus been shown, that there is an increasing demand for protamine and/or its derivatives. Although it is stated in the art, that protamine can be isolated from mammals, amphibians and fish, and although is has long been speculated in the art to provide protamine as a recombinant protein, no evidence of recombinant production has yet been published with the effect, that salmon (salmine®) and herring (clupeine®) protamine remain to be the only commercially available preparations to date.
Thus, the main object underlying the present invention is to overcome the drawbacks of the state of the art by providing a method for recombinantly producing bioactive protamine or functional fragments thereof enabling large-scale, cost-effective and reliable production of this interesting substance.